Device Preparation Using Condensed Nucleic Acid Particles

ABSTRACT

A method of sequencing a nucleic acid strand includes receiving particles having nucleic acid strands coupled to a polymer matrix, exposing the particles to a solution including a condensing agent, and applying the particles to a surface, the particles depositing on the surface.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application claims benefit of U.S. Provisional Application No.61/877,745, filed Sep. 13, 2013, which is incorporated herein byreference in its entirety.

This application claims benefit of U.S. Provisional Application No.62/020,292, filed Jul. 2, 2014, which is incorporated herein byreference in its entirety.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to systems and methods forpreparing devices for use in nucleic acid sequencing, particularly suchdevices that utilize nucleic acid containing p articles.

BACKGROUND

Sequencing of nucleic acid strands, particularly DNA, has becomeincreasingly important in advancing fields including medicine,agriculture, and biological research. However, conventional geltechniques for sequencing nucleic acid strands have proventime-consuming and expensive. More recent developments rely on thedeposition of nucleic acid containing samples on substrates. Dependingupon the sequencing technique, the sequence of the nucleic acid samplecan be determined by measuring ionic responses to nucleic acid additionor by measuring fluorescent emissions resulting from nucleic acidaddition.

However, such techniques that rely on the deposition of nucleic acidsamples on a substrate suffer from deficiencies caused by a failure ofsome nucleic acid strands to bind to the surface of the substrate orcaused by strands binding in close proximity to each other. Suchdeficiencies can lead to underutilization of the substrate, inaccuratedata, lost or incomplete samples, or other inaccuracies.

As such, an improved system and method for preparing a sequencing devicewould be desirable.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1 includes an illustration of an exemplary method for depositingnucleic acid samples.

FIG. 2 includes an illustration of an exemplary device for preparingnucleic acid particles and preparing a device for sequencing.

FIG. 3 includes images of beads or particles during exposure to hexaminecobalt.

FIG. 4, FIG. 5, FIG. 6, FIG. 7, and FIG. 8 include graph illustrationsof the size response of beads or particles to exposure to hexaminecobalt.

FIG. 9 includes a graph illustration of the dissociation kinetics ofnucleic acid conjugated beads or particles both with and withoutexposure to hexamine cobalt.

FIG. 10 and FIG. 11 include graph illustrations of the influence ofcondensing agents on bead size.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

In an embodiment, a method includes preparing a nucleic acid bead orparticle, condensing the nucleic acid bead, and depositing the condensednucleic acid bead on a sensor substrate. In another example, the methodincludes depositing a nucleic acid bead on the sensor substrate andcondensing the deposited nucleic acid bead. A nucleic acid bead orparticle includes a bead conjugated to one or more nucleic acid strands.Condensing the nucleic acid bead can include condensing nucleic acidsconjugated to the bead, shrinking the polymer of the bead, or acombination thereof, effectively reducing the size of the nucleic acidbead. After deposition, the condensed nucleic acid beads can be washed,which can expand the condensed nucleic acid beads. Subsequently, thesensor substrate with the deposited nucleic acid beads can be used fornucleic acid testing, such as sequencing, for example,sequencing-by-synthesis. Condensing the nucleic acid bead or particlecan include exposing the nucleic acid bead or particle to a condensingagent. The condensing reagent can condense aspects of a nucleic acidbead or particle, for example, condensing the nucleic acid, the polymermatrix of the particle, or a combination thereof. In an example, thecondensing agent includes a metal complex having a 3⁺ charge. Forexample, the metal complex can be a cobalt complex, such as acobalt-amine complex. In another example, the condensing agent includesconcentrated alkali or alkali-earth metal salts, such as a magnesiumsalt. In a further example, the condensing reagent includes a non-ionicpolymeric reagent, such as a polyethylene glycol based reagent.

In another embodiment, a system implements a method for preparing adevice for sequencing. The system can include a solution container, asample receiving port or sample chamber, and a mixer coupled to thesolution container and the sample receiving port or chamber. The mixercan be coupled to a substrate preparation unit. In an example, asolution in the solution container includes a condensing reagentincluding a condensing agent. Alternatively, the methods and processesdescribed herein can be performed manually or with the use of stirplates, vortexers, pipettes, centrifuges, or other bench equipment.

The sample receiving port or sample chamber is to receive nucleic acidbeads or particles, which have a polymeric matrix and a nucleic acidstrand coupled to the polymeric matrix. The condensing reagent and thenucleic acid particles are mixed, such as in the mixer. As a result, thenucleic acid strand or the polymer matrix condenses, resulting in aparticle with a reduced diameter or a greater density. The treatedparticles are transferred to a substrate preparation unit in which theparticles are deposited on the substrate. In a particular example, thesubstrate includes a layer that defines wells, and the particles aredeposited within wells. The substrate can be used for sequencing thenucleic acid strand of the bead or particle. In an example, thesubstrate is transferred to a sequencing device which performs functionsresulting in sequencing of the nucleic acid strand.

Alternatively, the nucleic acid beads can be applied to the sensorsubstrate and the condensing reagent applied over the sensor substrateafter the nucleic acid beads are deposited. In a further example, thenucleic acid beads can be treated with condensing agent prior todeposition, deposited, washed, and treated with condensing agentfollowing deposition and washing.

As illustrated in FIG. 1, a nucleic acid bead or particle 102 includes apolymer matrix 104 and nucleic acid strands 106. While the nucleic acidstrands 102 are illustrated as extending from a surface of the polymermatrix 104, the polymer matrix 104 can be porous, for example ahydrogel, and the nucleic acid strands 102 can be coupled to and extendthroughout the polymer matrix 104. The nucleic acid beads or particles102 can be treated to form treated nucleic acid beads or particles 108.The treated nucleic acid particle 108 can have a smaller diameter thanthe nucleic acid particle 102.

A surface component 116 includes a substrate 110, which includes a layer112 that defines wells 114. The treated nucleic acid particle 108 can bedeposited in the well 114. Subsequently, the treated nucleic acidparticle 108 can be washed while it remains in the well 114. Washing canresult in a nucleic acid particle with an increased diameter. Sequencingor other experimentation can be performed using the nucleic acid bead orparticle while it remains in the well 114.

In an example, the nucleic acid beads or particles 102 include thepolymer matrix 104 and one or more nucleic acid strands 106 coupled tothe polymer matrix 104. The polymer matrix 104 can be formed of ahydrophobic polymer or can be formed of a hydrophilic polymer, such as ahydrogel matrix. In particular, the polymer matrix 104 can include apolymer, such as a polysaccharide such as agarose, hyaluronan, ormethylcellulose; a polyoxyolefin such as polyoxybutylene,polyoxyethylene or polyethylene glycol, or polyoxypropylene; anacrylamide such as dimethylacrylamide, polyacrylamide,N,N-polydimethylacrylamide, poly(N-isopropylacrylamide),poly-N-hydroxyacrylamide, poly-N-hydroxyalkylacrylamide, or aminefunctional variants thereof; polyvinylpyrrolidone; polystyrene;silicone; poly(2-acrylamido-2-methyl-1-propanesulfonic acid); otheracrylate polymers; polyvinyl alcohol; copolymers or derivatives thereof;or any combination thereof. In a particular example, the polymer matrix104 can be formed of polystyrene. In another example, the polymer matrix104 is formed of polyoxyethylene. In a further example, the polymermatrix 104 is formed of an acrylamide, such as hydroxyalkylacrylamide,amine terminated acrylamide, or a derivative thereof.

For example, the polymer matrix 104 can be formed from monomersincluding a radically polymerizable monomer, such as a vinyl-basedmonomer. In an example, the monomer can include acrylamide, vinylacetate, hydroxyalkylmethacrylate, or any combination thereof. In aparticular example, the hydrophilic monomer is an acrylamide, such as anacrylamide including hydroxyl terminal groups, amino terminal groups,carboxyl terminal groups, or a combination thereof. In an example, thehydrophilic monomer is an aminoalkyl acrylamide, an acrylamidefunctionalized with an amine terminated polypropylene glycol (D,illustrated below), an acrylopiperazine (C, illustrated below), or acombination thereof. In another example, the acrylamide can be ahydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. In particular,the hydroxyalkyl acrylamide can includeN-tris(hydroxymethyl)methyl)acrylamide (A, illustrated below),N-(hydroxymethyl)acrylamide (B, illustrated below), or a combinationthereof. In a further example, a mixture of monomers, such as a mixtureof hydroxyalkyl acrylamide and amine functionalize acrylamide or amixture of acrylamide and amine functionalized acrylamide, can be used.In an example, the amine functionalize acrylamide can be included in aratio of hydroxyalkyl acrylamide:amine functionalized acrylamide oracrylamide:amine functionalized acrylamide in a range of 100:1 to 1:1,such as a range of 100:1 to 2:1, a range of 50:1 to 3:1, a range of 50:1to 5:1 or even a range of 50:1 to 10:1.

In a particular example, the polymer matrix 104 is a hydrogel beadsubstrate.

The polymer matrix 104 can include coupling sites to which a templatepolynucleotide can hybridize. The couplings sites can include theterminal groups, such as hydroxyl or amine terminal groups. For example,the coupling sites can each include a coupling oligonucleotidecomplementary to a section of a template polynucleotide. The templatepolynucleotide can include the target polynucleotide or segmentscomplementary to the target polynucleotide, in addition to segmentscomplementary to the coupling oligonucleotide.

The coupling oligonucleotide can be conjugated to the polymer matrix104. The polymer of a polymer matrix 104 can be activated to facilitateconjugation with a target analyte, such as an oligonucleotide orpolynucleotide. For example, functional groups on the polymer matrix 104can be enhanced to permit binding with target analytes or analytereceptors. In a particular example, functional groups of the hydrophilicpolymer can be modified with reagents capable of converting thehydrophilic polymer functional groups to reactive moieties that canundergo nucleophilic or electrophilic substitution. For example,hydroxyl groups on the substrate can be activated by replacing at leasta portion of the hydroxyl groups with a sulfonate group or chlorine.Exemplary sulfonate groups can be derived from tresyl, mesyl, tosyl, orfosyl chloride, or any combination thereof. Sulfonate can act to permitnucleophiles to replace the sulfonate. The sulfonate may further reactwith liberated chlorine to provide chlorinated groups that can be usedin a process to conjugate the particles. In another example, aminegroups on a substrate can be activated.

For example, target analyte or analyte receptors can bind to thehydrophilic polymer through nucleophilic substitution with the sulfonategroup. In particular example, target analyte receptors terminated with anucleophile, such as an amine or a thiol, can undergo nucleophilicsubstitution to replace the sulfonate groups on the surface of thepolymer matrix 104.

In another example, sulfonated polymer matrices can be further reactedwith mono- or multi-functional mono- or multi-nucleophilic reagents thatcan form an attachment to the particle while maintaining nucleophilicactivity for oligonucleotides comprising electrophilic groups, such asmaleimide. In addition, the residual nucleophilic activity can beconverted to electrophilic activity by attachment to reagents comprisingmulti-electrophilic groups, which are subsequently to attach tooligonucleotides comprising nucleophilic groups.

In another example, a monomer containing the functional group can beadded during the polymerization. The monomer can include, for example,an acrylamide containing a carboxylic acid, ester, halogen or otheramine reactive group. The ester group may be hydrolyzed before thereaction with an amine terminated oligonucleotide.

Other conjugation techniques include the use of monomers that compriseamines. The amine is a nucleophilic group that can be further modifiedwith amine reactive bi-functional bis-electrophilic reagents that yielda mono-functional electrophilic group subsequent to attachment to thebead or particle. Such an electrophilic group can be reacted witholigonucleotides having a nucleophilic group, such as an amine or thiol,causing attachment of the oligonucleotide by reaction with the vacantelectrophile.

If the polymer matrix is prepared from a combination of amino- andhydroxyl-acrylamides, the substrate can include a combination ofnucleophilic amino groups and neutral hydroxyl groups. The amino groupscan be modified with di-functional bis-electrophilic moieties, such as adi-isocyanate or bis-NHS ester, resulting in a hydrophilic particlereactive to nucleophiles. An exemplary bis-NHS ester includesbis-succinimidyl C2-C12 alkyl esters, such as bis-succinimidyl suberateor bis-succinimidyl glutarate.

Other activation chemistries include incorporating multiple steps toconvert a specified functional group to accommodate specific desiredlinkages. For example, a sulfonate modified hydroxyl group can beconverted into a nucleophilic group through several methods. In anexample, reaction of the sulfonate with azide anion yields an azidesubstituted hydrophilic polymer. The azide can be used directly toconjugate to an acetylene substituted biomolecule via “CLICK” chemistrythat can be performed with or without copper catalysis. Optionally, theazide can be converted to amine by, for example, catalytic reductionwith hydrogen or reduction with an organic phosphine. The resultingamine can then be converted to an electrophilic group with a variety ofreagents, such as di-isocyanates, bis-NHS esters, cyanuric chloride, ora combination thereof. In an example, using di-isocyanates yields a urealinkage between the polymer and a linker that results in a residualisocyanate group that is capable of reacting with an amino substitutedbiomolecule to yield a urea linkage between the linker and thebiomolecule. In another example, using bis-NHS esters yields an amidelinkage between the polymer and the linker and a residual NHS estergroup that is capable of reacting with an amino substituted biomoleculeto yield an amide linkage between the linker and the biomolecule. In afurther example, using cyanuric chloride yields an amino-triazinelinkage between the polymer and the linker and two residualchloro-triazine groups one of which is capable of reacting with an aminosubstituted biomolecule to yield an amino-triazine linkage between thelinker and the biomolecule. Other nucleophilic groups can beincorporated into the particle via sulfonate activation. For example,reaction of sulfonated particles with thiobenzoic acid anion andhydrolysis of the consequent thiobenzoate incorporates a thiol into theparticle which can be subsequently reacted with a maleimide substitutedbiomolecule to yield a thio-succinimide linkage to the biomolecule.Thiol can also be reacted with a bromo-acetyl group.

Alternatively, acrydite oligonucleotides can be used during thepolymerization to incorporate oligonucleotides. An exemplary acryditeoligonucleotide can include an ion-exchanged oligonucleotides.

The polymer matrix 104 can have a diameter in a range of 0.1 μm to 15μm. For example, the polymer matrix 104 can have a diameter in a rangeof 0.1 μm to 10 μm, such as a range of 0.1 μm to 5.0 μm, a range of 0.1μm to 3.0 μm, or a range of 0.1 μm to 1 μm. In a particular example, thecore 104 can have a diameter in a range of 0.1 μm to 0.8 μm, such as arange of 0.1 μm to 0.5 μm.

The nucleic acid strand 106 can have a length of at least 50 bases. Forexample, the nucleic acid strand can have a length of at least 100bases. In a particular example, the nucleic acid strand can have alength between 100 and 10,000 bases, such as between 100 and 8,000bases, between 100 and 5,000 bases, between 100 and 1,000 bases, orbetween 150 and 500 bases. The nucleic acid bead or particle 102 caninclude more than one nucleic acid strand bound to the polymer matrix104. In a particular example, the nucleic acid strands on a nucleic acidparticle are identical. For example, the particle can include at least100, such as at least 1000, or even at least 10,000 identical copies ofthe nucleic acid strand. In particular, the nucleic acid particle 102can include at least one 100,000 copies of the nucleic acid strand 106,such as at least 1 million copies of the nucleic acid strand. In anexample, the nucleic acid particle 102 includes not greater than 100million copies of the nucleic acid strand.

When mixed with a solution including a condensing agent, the nucleicacid strands or the polymer matrix condense. In an example, thecondensing agent includes a metal-complex. The metal-complex can have 3⁺charge and can be provided to the solution as a salt. For example, themetal complex salts can be a halide salts, such as a chloride salt oriodide salt. In particular, the metal complex includes cobalt, forexample, forming a cobalt organic complex, such as a cobalt-aminecomplex. In an example, the metal-complex can include hexamine cobalt.In another example, the metal-complex includes tris(ethylenediamine)cobalt. In a further example, the metal complex includes cobaltsepulchate.

In particular, the solution can include the metal-complex in aconcentration, such that when mixed with the sample, the concentrationof the metal-complex in the resulting solution is at least 1 μM, such asat least 10 μM, at least 20 μM or at least 50 μM. In an example, theconcentration can be in a range of 10 μM to 1 mM, such as a range of 20μM to 500 μM, or even a range of 20 μM to 350 μM. In an alternativeexample, the concentration is at least 200 μM, such as at least 500 μM,or at least 1 mM. For example, the concentration can be at least 4.7 mM,such as at least 9.8 mM, at least 14.9 mM, or even at least 19.7 mM. Inparticular, the concentration is not greater than 100 mM, such as notgreater than 50 mM. When expressed relative to the concentration of DNA,where the concentration of DNA is expressed in μg/ml, the ratio of theconcentration of the metal-complex to the concentration of DNA is atleast 0.5, such as at least 1.0, at least 1.5, at least 2.0, or even atleast 3.0, but not greater than 100.

Alternatively or additionally, the solution can include a condensingagent that influences the density of the polymer matrix 104 of the beador particle 102. For example, the solution can include an alcohol, suchas methanol, ethanol or isopropyl alcohol (IPA), which can influence thedensity of the polymer matrix 104 of the particle 102. In particular,the solution can include an alcohol, such as methanol, in aconcentration in a range of 0.1 vol. % to 60 vol. %, such as 0.1 vol. %to 50 vol. %, 0.1 vol. % to 30 vol. %, 0.1 vol. % to 20 vol. %, or even0.1 vol. % to 10 vol. %. For example, the concentration of alcoholwithin the solution can be in a range of 0.1 vol. % to 5 vol. %, such as1 vol. % to 5 vol. %. In another example, the solution can include analcohol, such as ethanol or IPA in a concentration in a range of 0.1vol. % to 60 vol. %, such as 1.0 vol. % to 60 vol. %, 5.0 vol. % to 60vol. %, 10 vol. % to 60 vol. %, or even 40 vol. % to 60 vol. %.

In another example, the condensing agent includes concentrated alkali oralkali-earth metal salts, such as halide salts. In an additionalexample, the condensing agent can include magnesium chloride, forexample, in a concentration in a range of 1 mM to 1M, such as a range of30 mM to 1M, a range of 50 mM to 1M, a range of 50 mM to 800 mM, or evena range of 50 mM to 500 mM.

The condensing agent can further be a non-ionic polymer, such as apolyethylene glycol based polymer In a particular example, thepolyethylene glycol based polymer has a molecular weight in a rangebetween 1000 and 100000, such as a range between 2000 and 20000, between5000 and 15000, or between 8000 and 12000. The non-ionic polymer can beincluded in a concentration in a range of 0.1 wt % to 20.0 wt %, such asa range of 0.5 wt % to 15.0 wt %, a range of 0.5 wt % to 10 wt %, or arange of 2.5 wt % to 7.5 wt %.

The remainder of the solution can include a buffered solution, such asbuffered saline solution. For example, the remainder of the solution caninclude a phosphate buffered saline solution. In particular, thesolution can include sodium or potassium halide salts, sodium orpotassium phosphate salts, and polysorbate. Such salts can be included,for example, in amounts in a range of 1 mM to 500 mM, such as 50 mM to350 mM, or even 150 mM to 250 mM. In an example, potassium chloride canbe included in a concentration in a range of 0.5 M to 2 M, such as arange of 0.8M to 1.5M, or even a range of 0.8M to 1.2M. In addition oralternatively, other buffering agents can be used, such as an aminebased buffering agent, e.g., tris(hydroxymethyl)aminomethane. Such anamine buffering agent can be used in a concentration in a range of 50 mMto 1M, such as a range of 100 mM to 1M, a range of 100 mM to 800 mM, oreven a range of 150 mM to 500 mM. Optionally, the solution can includeother ionic components, such as calcium or magnesium, derived fromsalts. Further, the solution can have a pH between 6 and 9, such asbetween 6.5 and 8.5, or between 7 and 8.5.

The solution can include a surfactant. For example, the surfactant canbe a non-ionic polymer surfactant, such as an ether of polyethyleneglycol, for example an octylphenyl ether of polyethylene glycol. Thenon-ionic polymer surfactant can be included in a range of 0.01% to1.0%, such as a range of 0.05% to 0.8%, a range of 0.05% to 0.5%, oreven a range of 0.08% to 0.15%. An exemplary surfactant is TritonX-100.

In an example, a condensing solution includes a condensing agent, suchas MgCl₂ in a range of 30 mM to 500 mM; a salt, such as KCl in a rangeof 0.8M to 1M; a buffering agent, such astris(hydroxymethyl)aminomethane in a range of 150 mM to 500 mM; and asurfactant, such as TritonX-100 in a range of 0.05% to 0.5%. The pH isin a range of 7 to 9.

In another example, the a condensing solution includes a combination ofcondensing agents, such as MgCl₂ in a range of 30 mM to 500 mM andpolyethylene glycol in a range of 0.5 wt % to 10.0 wt %. Thepolyethylene glycol can have a molecular weight in a range of 5000 to15000. The condensing solution can also include a salt, such as KCl in arange of 0.8M to 1M; a buffering agent, such astris(hydroxymethyl)aminomethane in a range of 150 mM to 500 mM; or asurfactant, such as TritonX-100 in a range of 0.05% to 0.5%. The pH isin a range of 7 to 9.

In response to exposure to the solution, the nucleic acid beads orparticles 102 including the nucleic acid strands 106 can decrease indiameter. For example, the bead or particle diameter can decrease by atleast 1% in response to exposure to the solution. In an example, thediameter decreases by at least 5%, such as at least 10%, at least 15%,or even at least 19%. In a particular example, the diameter decreases bynot greater than 75%. Further, the density of the bead or particle canincrease in response to exposure to a condensing agent. For example, thedensity can increase by at least 2%, such as at least 8%, at least 14%,at least 21%, or even at least 25%. In particular, the density increasesby not greater than 75%.

Despite condensation of the nucleic acid strands 106 or the polymermatrix 104 in response to exposure to the condensing solution orreagent, the nucleic acid strands after exposure to the solution canexhibit a similar enzyme dissociation constant when compared to such aconstant of the nucleic acid strands before exposure. For example, thenucleic acid strands after exposure can exhibit a similar dissociationconstant when in the presence of a polymerase when compared with thenucleic acid strands before exposure.

Returning to FIG. 1, following exposure to the solution, the treatednucleic acid particles 108 can be applied to a surface component 116 ofa sensor substrate, such as a sequencing device. For example, asuspension including the treated nucleic acid particles 108 can beapplied over a surface of the surface component 116, such as by flowingthe suspension across the surface, by foaming the suspension andapplying the foam across the surface, or by applying the suspension andcentrifuging the component 116. In a particular example, the surfaceincludes regions to which the particles are secured, immobilized orbound. For example, the surface can include treated areas that areresponsive to a surface agent on the beads or particles. In particular,the surface component 116 can include a pattern of treated areas, suchas patterns of metal deposition.

In another example, the surface component 116 includes a layer 112 thatdefines wells 114 into which the treated nucleic acid beads or particles108 can be deposited. Alternatively, the surface component 116 caninclude discrete sites, such as pits, grooves, channels, dimples, orother well-like sites. In a particular example, the surface component116 can define wells 114 that correspond to active circuits formeasuring ionic concentration, such as pH. Alternatively, the activecircuits can measure, heat, fluorescence, or phosphate concentration. Inanother example, the wells 114 can be deposited over transparent layersof the surface component 116 for receiving or transferringelectromagnetic radiation or fluorescent emissions.

The wells 114 can have an effective diameter of not greater than 10 μm,such as not greater than 7 μm, not greater than 4 μm, not greater than 3μm, not greater than 2 μm, not greater than 1.5 μm, not greater than 1.0μm, or even not greater than 0.8 μm. The effective diameter is thesquare root of the product of four times the cross-sectional areadivided by pi (i.e., d_(effective)=sqrt(4A/π)). In particular, theeffective diameter can be not greater than 1.0 μm or not greater than0.8 μm. The effective diameter can be at least 0.3 μm. Further, thewells 114 can have a depth of not greater than 10 μm, such as notgreater than 5 μm, not greater than 3 μm, not greater than 1.5 μm, oreven not greater than 1 μm. For example, the depth can be not greaterthan 500 nm, such as not greater than 200 nm, or even not greater than150 nm. The depth can be at least 100 nm.

The wells 114 can define a volume of not greater than 1 microliter, suchas not greater than 100 nanoliters, not greater than 10 nanoliter, ornot greater than 1 nanoliter. For example, the wells can define a volumeof not greater than 100 picoliters, such as not greater than 10picoliters, or even not greater than 1 picoliter. In particular, thewells can define a volume of not greater than 100 femtoliters, such asnot greater than 50 femtoliters, such as not greater than 20femtoliters, not greater than 10 femtoliters, not greater than 5femtoliters, not greater than 1 femtoliter, or even not greater than 0.6femtoliters, but can be at least 0.01 femtoliters.

Following deposition of the particles on surface, the surface component116 can be placed in a sequencing system. Optionally, the surfacecomponent 116 can be washed to remove the condensing agent prior toplacing it in the sequencing system or can be washed once placed in thesequencing system. In response, the nucleic acid bead or particle canincrease in diameter or the nucleic acid strands can expand from acondensed state. For example, the beads or particles can be washed witha buffered solution, such as a buffered saline solution. In particular,the solution can be a solution similar to the condensing agentcontaining solution, absent the condensing agent.

Alternatively, the nucleic acid bead can be loaded before exposure tothe condensing agent. For example, the nucleic acid beads can be appliedto a sensor substrate and a solution including the condensing agent canbe applied over the deposited nucleic acid beads. In a furtheralternative example, the nucleic acid bead can be treated with thecondensing agent before depositing, deposited, washed, and treated withthe condensing agent after being deposited.

Once placed in the sequencing system, the nucleic acid strands attachedto the particles that are deposited on the surface component can besequenced. In an example, sequencing can include measuring ionconcentration in response to nucleotide addition. In such an example, asolution including a single type of nucleotide is contacted with thenucleic acid strands and the sequencing system monitors for changes inion concentration, such as changes in pH. Subsequently, a solution witha different type of nucleotide can be applied to the surface and the ionconcentration again monitored. In such a manner, the addition ofindividual nucleotides can be monitored and detected so as to determinewhich nucleotides are attached and in what order. In an alternativeexample, sequencing can include measuring fluorescent emissions inresponse to nucleotide addition. Nucleotides can be fed sequentially inseparate solutions for each nucleotide or for nucleotides having adifferent fluorescent species, which fluoresce at different wavelengths,associated with each type of nucleotide, the nucleotides can be fed in acombined solution into the sequencing system.

Such a method can be implemented in a system, such as system 200illustrated in FIG. 2. For example, the system 200 can include asolution container 202. Further, the system 200 includes a sample portor sample receiving chamber 204. Both the solution container 202 and thesample portal or receiving chamber 204 feed into a mixer 206. The mixer206 can be an agitated mixer, an ultrasonic mixer, or an in-line mixerwithin tubing extending to a surface preparation unit 208. Inparticular, the mixing device 206 feeds treated particles received atthe sample port or chamber 204 to a surface component to be prepared foruse in the sequencing device. Alternatively, the solution container 202and sample port or receiving chamber 204 can feed into the surfacepreparation unit 208 without entering a mixer or being mixed.

The surface preparation unit 208 can include a chamber in which thesensor substrate forms a major surface and through which the solutionincluding a condensing agent and the nucleic acid beads or particlesincluding nucleic acid strands flow across the surface to facilitatedeposition of the beads or particles on the sensor substrate. In aparticular example, the sensor substrate can include a flow cell definedover a sensor surface including wells. The surface preparation unit canfurther include agitators, aspirators, centrifuges, pipetters, orvortexers to further enhance deposition of the beads or particles intothe wells of the sensor substrate. Alternatively, the process can beperformed manually using the above equipment.

As a result of the method, the particles can be deposited on the surfacecomponent to a desirable surface density. For example, such a method canprovide a surface component having an occupancy, defined as the percentof wells that include a DNA containing particle, of at least 60%, suchas at least 80%, at least 85%, at least 87%, or even at least 89%. Inparticular, the occupancy can be at least 90%, such as at least 93%, oreven at least 95%.

It is believed that an increase in density and a decrease in diameterassist with the concentrated deposition of particles on the surface.Following deposition, the particles can be washed either in the surfacepreparation device 208 or once placed in the sequencing system.

EXAMPLES Example 1

Particles formed from DNA containing polymeric particles (ION Spheres™following PCR amplification) are exposed to hexamine cobalt inconcentrations ranging from 300 μM to 20 mM. FIG. 3 illustrates changesin particle diameter in response to changing concentration of hexaminecobalt. Initially, the particles are in a phosphate buffer saline withTween-20 (PBST) solution (e.g., 1 liter aqueous solution including 8 gNaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, and 2 ml of tween-20).As the concentration of hexamine cobalt increases, the diameter of theparticle decreases. The particle increases in diameter following washingwith a PBST solution.

DNA containing particles are tested as prepared or in contact withpolymerase enzyme. As illustrated in FIG. 4, the diameter of the DNAcontaining particles decrease with increasing concentration of hexaminecobalt. As illustrated in FIG. 5, the diameter increases when placed incontact with an enzyme. When the particle in contact with the enzyme isexposed to hexamine cobalt (5 mM Co-complex), the diameter decreases andsubsequently increases when washed with a buffered solution (PBST).

FIG. 6, FIG. 7, FIG. 8 illustrates the response of DNA containingparticles formed using a nucleic acid library in contact with differentenzymes. In each case, the diameter of the DNA containing particlesincrease when contacted with the enzyme and subsequently decreases inresponse to increasing concentrations of hexamine cobalt. Further, thediameter of the particles increases when washed first and second timeswith a buffered solution and when contacted with nucleic acidphosphates.

Particles including FAM-labeled hairpins are tested to measuredissociation kinetics with an enzyme, Bst1. As illustrated in FIG. 9,the dissociation kinetics as a function of ionic strength is similar forsamples exposed to hexamine cobalt and samples not exposed to hexaminecobalt.

DNA containing particles having an average core diameter of 0.7 micronsare provided in both a suspension free of metal-complex and a suspensionincluding hexamine cobalt. Substrates defining wells having an effectivediameter of less than 1 micron are exposed to one of the twosuspensions. The substrates are washed with a PBST solution and observedto determine how many of the wells are occupied with a DNA containingparticle. Those substrates exposed to the suspension including DNAcontaining particles free of metal-complex exhibit an occupancy, definedas the percent of wells that include a DNA containing particle, of lessthan 60%. Those substrates exposed to the suspension including DNAcontaining particles and hexamine cobalt exhibit an occupancy of greaterthan 85%, some exhibiting an occupancy of greater than 90%.

Example 2

An aqueous condensing reagent can include 1M KCl, 230 mM MgCl₂, 200 mMtris-HCL, and 0.1% Triton X-100. The reagent can have a pH ofapproximately 8.0. When the condensing agent is used during loading of apolyacrylamide bead (available from Ion Torrent™) conjugated topolynucleotides onto a Proton I™ chip (available from ION Torrent™), ithas been found that magnesium salt enhances loading of beads into wells.While not limiting the solution to a particular theory, it is believedthat the solution causes condensation of a hydrogel polymer matrix.

Example 3

An aqueous condensing reagent includes condensing agents 100 mM MgCl₂and 5 wt % polyethylene glycol. The polyethylene glycol has an averagemolecular weight of 10000. The condensing solution also includes 0.7 MKCl and 200 mM tris(hydroxymethyl)aminomethane. The pH is 8.0.

Example 4

Hydrogel beads (ION Spheres™ available from ION Torrent) are treatedwith a coloring agent (SBYR) and are observed using microscopy forresponse to condensing agents including magnesium and polyethyleneglycol (PEG). Bead size is expressed as a ratio relative to the beadsize in a low ionic strength saline solution.

FIG. 10 illustrates the response of bead size to differentconcentrations of magnesium in the presence of 5% PEG. As illustrated,the relative size initially drops rapidly with increasing concentrationsof magnesium. The rate of decrease slows with increasing concentrationsof magnesium.

FIG. 11 illustrates the response of bead size to differentconcentrations of PEG, either in the presence of 100 mM MgCl₂ or 50 mMMgCl₂. As illustrated, the relative size decreases with increasingconcentration of PEG. Polyethylene glycol may create a difference inosmotic pressure between the hydrogel bead and the external solution,causing water to exit the bead. The PEG may not enter the bead, but mayremain external to the bead, leading to the difference in osmoticpressure.

In a first aspect, a method of sequencing a nucleic acid strand includesreceiving particles having a polymer matrix conjugated to nucleic acidstrands, exposing the particles to a solution including a condensingagent, and applying the particles to a surface, the particles depositingon the surface.

In an example of the first aspect, the condensing agent includes a metalcomplex having a 3+ charge. For example, the metal complex can includecobalt. In a further example of the first aspect and the above example,the metal complex is a metal-amine complex. For example, the metalcomplex can include hexamine cobalt. In another example, the metalcomplex includes tris(ethylenediamine) cobalt. In a further example, themetal complex includes cobalt sepulchrate. In an additional example, themetal complex is derived from a chloride salt. In an additional exampleof the first aspect and the above examples, the solution includes themetal complex in a concentration of at least 1 μM. For example, theconcentration can be at least 10 μM, such as at least 1 mM, at least 4.7mM, or even at least 14.9 mM. In particular, the concentration is notgreater than 100 mM, such as not greater than 50 mM.

In a further example of the first aspect and the above examples, thesurface includes a layer defining wells and wherein applying theparticles to the surface includes applying the particles into the wells.

In another example of the first aspect and the above examples, thecondensing agent includes a non-ionic polymer. For example, thenon-ionic polymer includes a polyethylene glycol based polymer. Thepolyethylene glycol based polymer can have a molecular weight in a rangeof 2000 to 20000. In an example, the non-ionic polymer surfactant isincluded in a concentration of 0.1% to 10.0% by weight.

In a further example, the condensing agent includes an alkali oralkali-earth salt. For example, the condensing agent includes apotassium salt in a concentration of 0.5M to 2.0M. In another example,the condensing agent includes a magnesium salt in a concentration of 30mM to 1.0M.

In an additional example of the first aspect and the above examples, thecondensing agent includes an alkali or alkali-earth metal salt and anon-ionic polymer surfactant.

In another example of the first aspect and the above examples, adiameter of the particle decreases by at least 1% in response to theexposure. For example, the diameter can decrease by at least 5%, such asat least 10%, at least 15%, or at least 19%. In particular, the diametercan decrease by not greater than 75%.

In a further example of the first aspect and the above examples, adensity of the particles increases by at least 2% in response to theexposure. For example, the density can increase by at least 8%, such asat least 14%, or at least 21%. In particular, the density can increaseby not greater than 75%.

In an additional example of the first aspect and the above examples, thenucleic acid strands exhibit a similar dissociation after exposure tothe nucleic acid strands before exposure.

In another example of the first aspect and the above examples, thepolymer matrix includes a polymer including agarose, polyoxybutylene,dimethylacrylamide, polyoxyethylene or polyethylene glycol,polyacrylamide, polyoxypropylene, N,N-polydimethylacrylamide,poly(N-isopropylacrylamide), polyvinylpyrrolidone,poly-N-hydroxyacrylamide, poly-N-hydroxyalkylacrylamide, polystyrene,copolymer or derivatives thereof, or any combination thereof.

In an additional example of the first aspect and the above examples, themethod further includes washing the surface to remove the condensingagent.

In a further example of the first aspect and the above examples, themethod further includes sequencing the nucleic acid strand. For example,sequencing can include measuring ion concentration in response tonucleotide addition. In another example, sequencing can includemeasuring radiation emissions in response to nucleotide addition.

In a second aspect, a method of preparing a surface includes applying asolution to a surface, the solution including particles and a condensingagent, the particles depositing onto the surface. The method alsoincludes washing the surface with a solution free of the metal complex.

In a third aspect, a device includes a solution container including asolution including a condensing agent, a sample receiving port toreceive particles including nucleic acid strands, and a mixer in fluidcommunication with the solution container and the sample receiving port.

In an example of the third aspect, the device further includes a surfacepreparation unit to prepare a surface component of a sequencing deviceand to receive treated particles from the mixer.

In a fourth aspect, an aqueous reagent solution includes a magnesiumsalt in a range of 30 mM to 500 mM; a potassium salt in a range of 0.8 Mto 1.0 M; a buffering agent in a range of 150 mM to 500 mM; and asurfactant in a range of 0.05% to 0.5%.

In an example of the fourth aspect, the pH is in a range of 7 to 9. Inanother example of the fourth aspect and the above examples, thebuffering agent includes tris(hydroxymethyl)aminomethane. In a furtherexample of the fourth aspect and the above examples, the surfactantincludes a polyethylene glycol based surfactant.

In a fifth aspect, an aqueous reagent solution includes a magnesium saltin a range of 30 mM to 500 mM; polyethylene glycol in a range of 0.5 wt% to 10.0 wt %; a potassium salt in a range of 0.8 M to 1.0 M; and abuffering agent in a range of 150 mM to 500 mM.

In an example of the fifth aspect, the pH is in a range of 7 to 9. Inanother example of the fifth aspect and the above examples, thebuffering agent includes tris(hydroxymethyl)aminomethane.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. A method of sequencing a nucleic acid strand, themethod comprising: receiving particles having a polymer matrixconjugated to nucleic acid strands; exposing the particles to a solutionincluding a condensing agent; and applying the particles to a surface,the particles depositing on the surface.
 2. The method of claim 1,wherein the condensing agent includes a metal complex having a 3+charge.
 3. The method of claim 2, wherein the metal complex comprisescobalt.
 4. The method of claim 2, wherein the metal complex is ametal-amine complex.
 5. The method of claim 2, wherein the metal complexincludes hexamine cobalt.
 6. The method of claim 2, wherein the solutionincludes the metal complex in a concentration of at least 1 μM.
 7. Themethod of claim 1, wherein the condensing agent includes a non-ionicpolymer.
 8. The method of claim 7, wherein the non-ionic polymerincludes a polyethylene glycol based polymer.
 9. The method of claim 8,wherein the polyethylene glycol based polymer has a molecular weight ina range of 2000 to
 20000. 10. The method of claim 7, wherein thenon-ionic polymer is included in a concentration of 0.1% to 10.0% byweight.
 11. The method of claim 1, wherein the condensing agent includesan alkali or alkali-earth salt.
 12. The method of claim 11, wherein thecondensing agent includes a magnesium salt in a concentration of 30 mMto 1.0M.
 13. The method of claim 1, wherein the condensing agentincludes an alkali or alkali-earth metal salt and a non-ionic polymer.14. The method of claim 1, wherein the surface includes a layer definingwells and wherein applying the particles to the surface includesapplying the particles into the wells.
 15. The method of claim 1,wherein a diameter of the particle decreases by at least 1% and notgreater than 75% in response to the exposure.
 16. The method of claim 1,wherein a density of the particles increases by at least 2% and notgreater than 75% in response to the exposure.
 17. The method of claim 1,wherein the nucleic acid strands exhibit a similar dissociation afterexposure to the nucleic acid strands before exposure.
 18. The method ofclaim 1, wherein the polymer matrix comprises a polymer comprisingagarose, polyoxybutylene, dimethylacrylamide, polyoxyethylene orpolyethylene glycol, polyacrylamide, polyoxypropylene,N,N-polydimethylacrylamide, poly(N-isopropylacrylamide),polyvinylpyrrolidone, poly-N-hydroxyacrylamide,poly-N-hydroxyalkylacrylamide, polystyrene, copolymer or derivativesthereof, or any combination thereof.
 19. The method of claim 1, furthercomprising: washing the surface to remove the condensing agent.
 20. Themethod of claims 1, further comprising: sequencing the nucleic acidstrand.